Predictive Role of Circulating Immune Cell Subtypes Early after Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Acute Leukemia

BACKGROUND AND OBJECTIVES: Cells of innate immunity normally recover in the first weeks to months after allogenenic hematopoietic stem cell transplantation (allo-HSCT). Their relevance in terms of graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect is largely unknown. The predictive role of early recovery in the immune cells on acute GVHD and GVL effect after allo-HSCT was investigated in patients with acute leukemia who achieved the first complete remission. METHODS: Peripheral blood samples were taken at the median of 14 days (range, 12~29 days) after allo-HSCT. A cohort including 119 samples and characteristics of patients were analyzed. Immune cell populations were identified by flow cytometry. RESULTS: The median age was 49.0 years (range, 21~69) at transplantation. Univariate analysis showed that age less than 40 years old, lower frequencies of CD8+ T cells, invariant natural killer T (iNKT) cells, monocytic myeloid derived suppressor cells (M-MDSCs) and higher frequency of immature MDSCs were associated with occurrence of grade III–IV acute GVHD. Multivariate analyses showed that iNKT cells (hazard ratio (HR), 0.453, 95% CI, 0.091~0.844, p=0.024) and M-MDSCs (HR, 0.271, 95% CI, 0.078~0.937, p=0.039) were independent factors. Combination of higher frequencies of both cell subsets was associated with lower incidence of grade III–IV acute GVHD, whereas patients with lower frequency of iNKT cells and higher frequency of M-MDSCs showed significant higher probability of relapse. CONCLUSIONS: iNKT cells and M-MDSCs could be relevant cell biomarkers for predicting acute GVHD and/or relapse in acute leukemia patients treated with allo-HSCT.

125 cases of children with epilepsy peripheral blood B lymphocytes ,NK cells and NKT cells in the detection and analysis / 国际检验医学杂志

Objective To investigate the changes and clinical significance of CD19 ,CD20 ,CD3-CD16+ CD56+ (NK cells) and CD3+CD16+CD56+ (NKT cells) in the children with epilepsy .Methods 125 cases of children with epilepsy children confirmed by Pediatric neurology specialists in Zhongshan People's Hospital from January 2013 to March 2016 were selected as epilepsy group , while 125 cases of healthy children were selected as control group ,and the levels of CD19 ,CD20 ,NK cells and NKT cells in periph-eral blood of two groups were detected by flow cytometry .Results CD19 ,CD20 ,NK cells and NKT cells levels in epilepsy group were higher than those in control group ,and the difference was statistically significant (P<0 .05) .For ROC curve analysis ,CD19 optimal cutoff value of 9 .4 ,the area under the curve was 90 .3% ,with 91 .7% sensitivity .CD20 optimal cutoff value of 7 .75 ,the ar-ea under the curve was 87 .3% ,with 91 .7% sensitivity .NK cells optimal cutoff value of 8 .55 ,the area under the curve was 80 .5% , with 95 .8% sensitivity .NKT cells optimal cutoff value of 0 .35 ,the area under the curve was 76 .9% ,with 91 .7% sensitivity .Con-clusion CD19 ,CD20 ,NK cells and NKT cells can be used as important indexes for monitoring immune conditions in children with epilepsy .

Lymphocyte subsets immune characteristics in hand foot and mouth disease：Its detection and clinical value / 上海预防医学

Objective] To summarize the immune characteristics in hand foot and mouth disease (HFMD) children with regards to peripheral blood T lymphocyte , B lymphocyte, NK cell and NKT cell, and to discuss its mechanism in the development of HFMD and their detection and clinical value . [ Methods] By means of flow cytometry , 152 children with HFMD and 45 healthy children were detected for the percentage of T lymphocyte , B lymphocyte, NK cell, NKT cell in peripheral blood . [ Results] The percentage of CD3 +,CD3 +CD4 +, CD3 +CD8 +, NK cells and ratio of CD4 +/CD8 +of HFMD acute phase group were lower than those in healthy group , while NKT cells, B cells were higher.The percentage of CD3 +, CD3 +CD4 +, CD3 +CD8 +, NKT cells and CD4 +/CD8 +ratio of HFMD acute phase were lower than those of recovery phase , while B cells and NK cells were higher .The percentage of CD 3 +cells, CD3 +CD4 +cells, CD3 +CD8 +cells, NKT cells and the ratio of CD 4 +/CD8 +in severe HFMD group were lower than those in mild group , while B cells and NK cells were higher . [ Conclusion] The immune function of children with HFMD proved disordered;CD3 +cells, CD3 +CD4 +cells and B cells in peripheral lympho-cytes were increased during the acute phase of HFMD;NKT cells increased during recovery phase of HFMD.We conclude that lymphocyte subsets detection can be used as an index of prognosis of HFMD in children .

Changes of expression levels of NK,NKT cells and their receptors in peripheral blood of human and its correlation with rheumatoid arthritis / 中国免疫学杂志

Objective:To explore the changes of NK and NKT cells and the expression levels of their activated, inhibitory receptors in the peripheral blood of patients with newly diagnosed rheumatoid arthritis ( RA), and to reveal the potiential role of NK and NKT cells played in the pathogenesis of RA.Methods:32 patients with new onset RA and 15 healthy controls were recruited.Activated and inhibitory NK and NKT cells in peripheral blood were quantified by flow cytometry.The frequency of spontaneous and stimulated IFN-γ+NK and NKT cells and CD107aγ+NK cells were examined.Finally,the potential relationship between the frequency of NK and NKT cells subsets and clinical indexes were analyzed.Results:The frequencies of NK cells in peripheral blood in RA patients were sig-nificantly lower than those in the controls ( P=0.026 ).The frequencies of NKG2D+, NKP46+activated NK cells and NKG2C+, NKG2D+,NKP46+activated NKT cells in RA patients were significantly higher than those in the controls (P=0.011,P=0.010,and P<0.001,P=0.032,P=0.001,respectively),whereas the frequencies of KIR2DL3+,KIR3DL1+and NKG2A+inhibitory NK cells and KIR2DL3+,NKG2A+inhibitory NKT cells in RA patients were significantly lower (P=0.002,P=0.002,P=0.014,and P=0.027,P=0.002,respectively).Moreover, the frequencies of stimulated IFN-γ+NK cells and IFN-γ+NKT cells, spontaneous and stimulated CD107 aγ+NK cells in RA patients were significantly higher than that in the controls ( P=0.037, P=0.004 and P=0.001, P=0.001, respectively).Furthermore,the frequencies of NK cells,NKG2Aγ+and KIR2DL3γ+inhibitory NK cells were correlated significantly with the values of DAS28 in RA patients (r=0.357,P=0.045;r=0.399,P=0.024;r=0.468,P=0.021,respectively).Conclusion:Lower frequencies of NK cells, higher frequencies of activated NK cells and activated NKT cells, lower frequencies of inhibitory NK cells and inhibitory NKT cells, and higher NK cell activity may induce the autoimmune reaction involved in the pathogenesis of RA.

MicroRNA-150 regulates differentiation of NK and NKT cells through decre-asing CD122 expression / 中国免疫学杂志

To explore the effects of microRNA-150 deletion on the development and homeostasis of regulatory T cells (Treg),γδT cells,NK and NKT cells.Methods:microRNA-150 knockout mice were used and microRNA-150 expression was detected by Real-time PCR.The numbers of Treg ,γδT,NK and NKT cells in the thymus and spleen of normal control and microRNA-150 knockout mice were detected by Flow cytometry.Cell apoptosis was detected by Annexin V staining , and cell proliferation was detected by 5-Bromo-2-deoxyUridine ( Brdu ) incorporation.Results: microRNA-150 deletion did not affect the development and homeostasis of regulatory T cells (Treg) andγδT cells.However,microRNA-150 deletion resulted in a significant reduction of the NK and thymic NKT cell number.In addition, microRNA-150 deleted NK and NKT cells showed an arrested developmental and maturational phenotype with a reduced expression of NK 1.1 and CD122.Moreover , cell apoptosis was significantly increased in microRNA-150 deleted NK and thymic NKT cells ,while a lower cell proliferation rate was shown in the microRNA-150 deleted NK but not NKT cells.Conclusion: CD122 may play an important role in the development and homeostasis of mouse NK and NKT cells regulated by microRNA-150.

B Cells Promote Th1- Skewed NKT Cell Response by CD1d-TCR Interaction

CD1d expressing dendritic cells (DCs) are good glyco-lipid antigen presenting cells for NKT cells. However, resting B cells are very weak stimulators for NKT cells. Although alpha-galactosylceramide (alpha-GalCer) loaded B cells can activate NKT cells, it is not well defined whether B cells interfere NKT cell stimulating activity of DCs. Unexpectedly, we found in this study that B cells can promote Th1-skewed NKT cell response, which means a increased level of IFN-gamma by NKT cells, concomitant with a decreased level of IL-4, in the circumstance of co-culture of DCs and B Cells. Remarkably, the response promoted by B cells was dependent on CD1d expression of B cells.

Comparison of Invariant NKT Cells with Conventional T Cells by Using Gene Set Enrichment Analysis (GSEA)

BACKGROUND: Invariant Natural killer T (iNKT) cells, a distinct subset of CD1d-restricted T cells with invariant Valphabeta TCR, functionally bridge innate and adaptive immunity. While iNKT cells share features with conventional T cells in some functional aspects, they simultaneously produce large amount of Th1 and Th2 cytokines upon T-cell receptor (TCR) ligation. However, gene expression pattern in two types of cells has not been well characterized. METHODS: we performed comparative microarray analyses of gene expression in murine iNKT cells and conventional CD4+CD25-gammadeltaTCR- T cells by using Gene Set Enrichment Analysis (GSEA) method. RESULTS: Here, we describe profound differences in gene expression pattern between iNKT cells and conventional CD4+CD25-gammadeltaTCR- T cells. CONCLUSION: Our results provide new insights into the functional competence of iNKT cells and a better understanding of their various roles during immune responses.

Clinical significance of detecting the NKT cell content and its surface receptors NKG2A and NKG2D in the peripheral blood of patients with esophageal carcinoma / 肿瘤研究与临床

Objective NKT cells are very important as a kind of non-specific immune cells. Much attention in antitumor significance has been received in the study of its effect on malignant diseases. The aim of this study was to detect the expression of NKT cells and its CD+8 NKT subsets in peripheral blood of esophageal patients and normal person, and to analyze the changes in the expression of NKG2D and NKG2A receptorsand its clinical pathological factors. Methods By flow cytometric analysis, 53 patients with esophageal carcinoma and 39 normal controls were analysed for peripheral blood of NKT cells and CD+8 NKT subsets, and the expression of NKT cells NKG2A and expression of NKG2D receptor. The clinical pathological factors were collected for the comparative analysis. Results Compared with the normal control group, the expression of NKT cells in peripheral blood of esophageal patients increased [(4.32±0.73) %, (5.97±1.29) %] (t =3.562, P <0.01), and the expression level of its surface receptor NKG2D reduced [(17.56±5.92) %, (15.12±1.56) %] (t =3.892, P <0.05), but the express levels of NKG2A [(4.02±1.41) %, (5.99±4.59) %] in creased (r = 4.015, P <0.05), those expression change with the development of the esophageal cancer. Conclusion The increased expression of NKT cells and CD+8 NKT subsets in the peripheral blood of patients with esophageal carcinoma refletcs that the immune feedback of patients' antineoplastic effect is strengthened. The decresed expression of the active receptor NKG2D and the increased expression of the inhibitory receptor NKG2A on NKT cells might be one of mechanisms leading to the reduction of NKT cell activity and immune escape of patients with esophageal carcinoma. The changes of surface receptors of NKT cells may be associated with the development of the esophageal cancer.

Analysis of NKT cells and related cytokines of peripheral blood in primary biliary cirrhosis animal model / 中华检验医学杂志

Objective To investigate the cell phenotype for T cells in polyI: C induced primary biliary cirrhosis (PBC)animal model.Methods 20 female C57BI/6 mice,8 weeks old,were divided into model group and control group randomly. Mice in model group and control group were injected with polyI:C at a dose of 5 ms/ks and PBS,respectively.All mice were acrificed after 16 weeks after injection, and the sections of liver specimen were subjected to hematoxylin and eosin(H.E) staining.Serum AMA and ALP were detected.CD4+,CDs8+ and NKT cells in peripheral blood were determined by flow cytometry.The level of serum IL-4 and IFN-γ were assayed by EUSA.Results PBC mouse model was developed 16 weeks fter polyI: C injection. Infiltration of lymphocytes in portal area,positive serum AMA and high level of serum ALP were observed.The ratios of CD4+ T cells in model group and control group were(25.45±11.12)% and (26.72±0.63)%,respectively(t=0.314,P>0.05).The ratios of CDs+T cells in two groups were (18.3±0.91)% and (17.8±0.58)%,espectively(t=0.226,P>0.05).No significant change Was found for CD;and CDs+T cells in mice of both groups.However,NKT cells in peripheral blood of two groups were(11.56±5.09)% and (1.26±0.53)%,respectively(t=9.504,P<0.01).The number of NKT cells in model group was more than that of control group significantly.Simultaneously,serum L-4 and IFN·γ in mice of model group were also higher than that of control group.IL4 in senlm of two groups were (22.19±2.31)pg/ml and(8.72±0.87)pg/ml,respectively(t=58.06,P<0.01).IFN-γ in serum of two groups were(3.34±0.76)ng/ml and(1.14±0.21)ng/ml,respectively(t=23.31,P<0.01).Conclusions NKT cells increase greatly in eripheral blood of polyI:C induced PBC mouse model.NKT cells may play a critical role in the pathogenesis of PBC.

The study on cytotoxicity of cytokines produced by the activated human NKT cells on neuroblastoma / 소아과

PURPOSE: alpha-Galactosylceramide (alpha-GalCer)-stimulated human Valpha24 natural killer T (NKT) cells exert antitumor activity against some leukemia in a CD1d dependent and TCR-mediated manner, but could not kill CD1d-negative neuroblastoma (NB) cells. There are few reports about the direct antitumor effect of highly secreted cytokines by these cells on activation. In this study, using a cell-free supernatant (SPN) collected from plate bound hCD1d/alphaGalCer tetramers-stimulated NKT cells, we examined whether they could be helpful in the immunotherapeutic treatment of NB. METHODS: Cells were cultured in IMDM. The cytokines produced by NKT cells were measured with Cytometric Bead Array (CBA) analysis. Cell viability was evaluated by calcein-AM fluorescence with digital image microscopy scanning (DIMSCAN). The percentage of specific apoptosis was calculated by flow cytometric detection of apoptosis using annexin V and 7-AAD. RESULTS: The activated NKT cells secreted high levels of IL-2, INF-gamma, TNF-alpha. The SPN was significantly cytotoxic against four out of eight tested NB cell lines, through mainly apoptosis as evidenced by annexin-V staining and inhibition with the pretreatment of pancaspase blocker. This apoptosis was significantly inhibited when anti-TNF-alpha and anti-IFN-gamma neutralizing mAbs were used separately and it was completely abolished when the two mAbs were combined. CONCLUSION: IFN-gamma and TNF-alpha produced by NKT cells could exert synergistically direct anti-tumor activity through apoptosis on some NB cell lines.

Alteration of Innate Immune T and B Cells in the NC/Nga Mouse

BACKGROUND: Millions of people in the world are suffering from atopic dermatitis (AD), which is a chronic inflammatory skin disease triggered by Th2 immune responses. The NC/Nga mouse is the most extensively studied animal model of AD. Like human AD, NC/Nga mice demonstrate increased levels of IgE, a hallmark of Th2 immune responses. Adaptive immunity cannot be generated without help of innate immunity. Especially natural killer T (NKT) cells and marginal zone B (MZB) cells have been known to play important roles in linking innate immunity to adaptive immunity. METHODS: Through flow cytometric analysis and ELISA assay, we investigated whether these lymphocytes might be altered in number in NC/Nga mice. RESULTS: Our data demonstrated that the number of NKT cells was reduced in NC/Nga mice and IFNgamma production by NKT cells upon alpha-GalCer stimulation decreased to the levels of CD1d KO mice lacking in NKT cells. However, reduction of NKT cells in NC/Nga mice was not due to CD1d expression, which was normal in the thymus. Interestingly, there was a significant increase of CD1d(high)B220+ cells in the spleen of NC/Nga mice. Further, we confirmed that CD1d(high)B220+ cells are B cells, not dendritic cells. These CD1d(high)B220+ B cells show IgM(high)CD21(high)CD23low, a characteristic phenotype of MZB cells. CONCLUSION: We provide the evidence that there are decreased activities of NKT cells and increased number of MZB cells in the NC/Nga mice. Our findings may thus explain why NC/Nga mice are susceptible to AD.

The Number of NKT and NK Cells in Patients with Systemic Lupus Erythematosus is Decreased: Association with the Pathogenesis for SLE / 대한신장학회잡지

BACKGROUND: Systemic lupus erythematosus (SLE) is one of chronic autoimmune diseases of which the central pathophysiologic derangement has not been yet established. Recently, it has been suggested that immune-regulatory cells might affect the development of autoimmune diseases such as SLE and RA. NKT cells were reported to be strong candidate for regulatory cells to regulate immune responses in vivo. To elucidate the roles of immune regulatory cells in the pathogenesis of SLE, we investigated the fractional distribution and functional status of NK and NKT cells in peripheral blood mononuclear cells (PBMC) of SLE patients and healthy volunteers. METHODS: Twenty-two SLE patients and 18 age-matched healthy volunteers were included in this study. The analysis for NK and NKT cells fraction in PBMCs of patients and normal controls were performed by flow cytometric analysis. In addition, to explore the functional status of these cells in SLE patients, we stimulated PBMCs using phorbol ester and ionomycin and measured cytoplasmic IL-4 and IFN-gamma by flow cytometry. RESULTS: The number (percentage) of NK cells was lower in SLE patients (CD3-CD56+: 4.93+/-1.30%, CD3-CD94+: 4.03+/-1.00%) than in controls (11.28+/-1.77%, 8.15+/-1.40%; p< 0.01, respectively). Peripheral NK cell numbers negatively correlated with anti-dsDNA Ab levels (r=-0.431, p< 0.05) and ESR (r= -0.475, p< 0.05). However, the percentage of these cells was not correlated with renal activity or corticosteroid doses. SLE patients showed, compared with controls, significantly decreased numbers of NKT cells (CD3+CD56+: 1.79+/-0.42% vs 5.04+/-0.44%, CD3+CD94+: 1.21+/-0.27% vs 4.39+/-0.45%; p< 0.01, respectively). The cytoplasmic expression of IL-4 and IFN-gamma in NK and NKT cells of SLE patients stimulated using phorbol ester and ionomycin were almost similar to those of normal controls, suggesting the NKT cells from SLE patients are functionally intact. CONCLUSION: Our results suggested that the decreased numbers of immune regulatory cells were associated with the immune dysregulation of SLE patients. The cellular replacement of NKT cells may be one of useful therapeutic approaches for autoimmune diseases such as SLE.

A comparative study on the biological characteristics of V?24 NKT cells and CD3~+CD56~+CIK cells from human peripheral blood / 中国输血杂志

Objective To confirm the difference in the biological characteristics between V?24 natural killer T cells(NKT) and the cytokine-induced killer cell(CIK).Methods V?24 NKT cells and CIK cells were expanded from human peripheral blood mononuclear cells.Purified TCRV?24~+ NKT cells and CD3~+CD56~+CIK cells were obtained by using Dynal beads.The phenotype and the levels of cytokine and necrosis-related factors expression on the purified NKT cells and CIK cells were determined by flow cytometry.The cytotoxicity of the purified NKT cells and CIK cells were measured by means of DIOC18 staining and flow cytometry.Results The proportions of TCRV?24~+V?11~+NKT cells and CD3~+CD56~+CIK cells were elevated up to 90% after purification.The majority of V?24 NKT cells were CD4~+ and DN NKT cells.They demonstrated high levels of expression of TCRV?24,V?11,CD3 and CD161 and a low level of expression of CD56,but most purified CD3~+CD56~+CIK cells were CD8~+ T cells,with high levels of CD3 and CD56 expression,low level CD161 expression and little TCRV?24 and V?11expression.After antigen stimulation,the purified CD3~+CD56~+CIK cells showed higher levels of expression of IFN-?,TNF-?,Perforin,FasL and TRAIL,compared to the NKT cells.CD3~+CD56~+CIK cells secreted little IL-4,whereas,V?24~+NKT cells secreted high level of IL-4.In addition,the cytotoxic effect of the CD3~+CD56~+CIK cells on tumor cell lines K562,U937 and Jurkat were more intense than that of the NKT cells.Conclusion It is evident that TCRV?24~+ NKT cells and CD3~+CD56~+CIK cells differ absolutely in many ways and might play different roles in anti-tumor immunity and immune regulation.

Progresses in development and function of NKT cells / 中国免疫学杂志

Natural killer T (NKT) cells,a subset of lymphocytes that bridge innate and adaptive immune systems,involve in processes of infection immunity,tumor immunity,transplantation immunity and autoimmunity.A significant progress has been made in the mechanisms of origin,selection,differentiation and maturation of NKT cells.However,some viewpoints are still controversial,and need to be further intensively investigated.The potential therapeutic applications of functional NKT cells have been suggested in the prevention and the treatment of various diseases.